Isolation, Selection and Identification of Antagonistic Marine Bacteria and Safety

Assessment for their Potential Application as Probiotics

Abstract
Antimicrobial resistance burden and hazard associated with chemical treatment of infections
demanded for new antimicrobial natural alternative. Oceanic hunt by marine organism enforces to
produce contemporary and novel strategies to compete, survive and reproduce their population.
This fact facilitates to find new solutions to control pathogenic bacteria. In this study we have
isolated 272 marine bacteria among them 136 (50 %) were antagonistic to at least one of the four
pathogenic strains Listeria monocytogenes, Vibrio cholerae, E. coli and S. aureus. Only two strains
exhibited antibacterial activity against all four test strains, which were identified by 16S rDNA
sequencing as Brevibacterium halotolerans DK1-SA11 and Vibrio kanaloae DK6-SH8.
Antagonistic activity and tolerance assessment showed that strain DK1-SA11 has broader
spectrum of antagonistic activity, including superbug MRSA and pathogenic yeast Candida
albicans, and can tolerate boiling temperature and pH 2-12, as compare with strain DK6-SH8.

Phenotypic characterization by macroscopic and microscopic studies, biochemical

characterization by API kits ( API 20E, API CORENY, API 50CHB and API ZYM), and
genotypic classification by DNA recA, gyrA and gyrB genes phylogenetic analysis revealed that,
strain DK1-SA11 is a novel strain and does not belongs to genus Brevibacterium. The genotypic
data confirmed that strain DK-SA11 belongs to specie cluster of Bacillus subtilis subsp. spizizenii
with 96-99.9 % similarity with existing data of DNA sequences.

Probiosis characteristics, safety and tolerance assessment of marine isolate DK1-SA11

revealed that, strain DK1-SA11 can produce alkaline phosphatase, esterase (C-4), esterase lipase
(C-8), lipase (C-14), leucine arylamidase, valine arylamidase, acid phosphatase,
phosphohydrolase, -glucosidase and -glucosidase with variable concentrations which was
quantified by API ZYM kit assay. Gastrointestinal track tolerance revealed that there was no
significant change (P<0.05) in bacterial counts after 4 h at pH 2.5, and MIC of conjugated bile
acids taurodeoxycholic acid and nonconjugated salt deoxycholic acid were 1.0 mM and 0.25
mM, respectively. Strain DK1-SA11 does not produce non-hemolytic and hemolysin BL
enterotoxins and negative for entFM, bceT, cytK, Nhe and Hbl toxin genes. Moreover, strain is
non-hemolytic and negative for plasmids and lecithinase production. Additionally strain DK1-
SA11 was sensitive to all antibiotics listed in European food safety association standards for live
bacteria usage guideline for animals and human consumptions. Acute and chronic studies
confirmed that there was neither death, nor any treatment related health effects on mice. The oral
LD50 of strain DK1-SA11 was greater than 2 1011 cfu. Acute toxicity results on zebra fish (Danio
rerio) revealed that strain has neither fatal nor visible toxic effect to fish and the lethal
concentration (LC50) of DK1-SA11 to the fish was > 108 cfu.mL-1.

Growth optimization of Marine isolate DK1-SA11 revealed that, among all tested media,
marine broth 2216E was found to be the best media for bacterial growth and antibacterial
compounds production. Moreover, pH-7, Incubation temperature 25 C with 180 rpm for 60-72
h was optimized conditions. Out of 49 different carbohydrates D-mannose increases the
antibacterial activity by 33.3% and D-arabitol decreases antimicrobial activity by 44.4%. Cold
storage below -20 C of crude CFS showed activity even after three months; whereas after boiling
for 10 min CFS was 100% active. CFS loses its 100% antimicrobial activity after lipase, trypsin
and papain enzymatic treatments.

Analysis and compound profiling of strain DK1-SA11 have shown that maximum amount of
antibacterial compounds could be extracted from butanol extraction. Thin layer chromatography ,
high performance liquid chromatography and mass spectrometry data revealed that all three kind
of lipopeptides surfactin, iturin and fengycin are being produce by strain DK1-SA11 which have
similar mass peaks reported before. Taken together all our data we conclude that strain DK1-SA11
is a novel marine Bacillus which is qualified for its application as probiotics. Moreover, strain
DK1-S11 is a putative source of several antimicrobial compounds with broad spectrum of
bioactivity.
Keywords: Marine Bacteria; Broad spectrum; Antagonistic; Probiotics; Bacillus subtilis subsp.
spizizenii